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Vaccine 22 (2003) 287292

Long-term persistence of immunity after immunisation withHaemophilus influenzae type b conjugate vaccine

P. Helena Mkel a, Helena Kyhty a,, Tuija Leino a, Kari Auranen a,b,Heikki Peltola c, Nina Ekstrm a, Juhani Eskola a,1

a National Public Health Institute, Mannerheimintie 166, FIN-00300 Helsinki, Finlandb Rolf Nevanlinna Institute, University of Helsinki, PL 4, 00014 Helsingin yliopisto, Finland

c Hospital for Children and Adolescents, University of Helsinki, PL 281, 00029HUS Helsinki, Finland

Received 2 January 2002; received in revised form 10 May 2002

Abstract

Although Haemophilus influenzae type b (Hib) conjugate vaccines, after licensure in 1987, are now recommended for world-wide use,the duration of protective immunity afforded by them is not known. We therefore assessed the immunogenity at 910 years of age in37 children who had received the first Hib conjugate, PRP-D, in infancy (the Hib-conjugate group) and were now given a dose of Hibpolysaccharide (PS) as a test vaccine. The anti-Hib PS antibodies (Hib-ab) were measured before and after this test vaccination, and thevalues compared to those in 37 control children who had not previously received any Hib vaccine and in 13 children who had receivedHib PS vaccine in infancy (the Hib-PS group). Prior to the test vaccination, the Hib-ab concentrations in the Hib-conjugate group were3.6-fold higher than in the control group. After the test vaccination, the Hib-conjugate group had higher total Hib-ab concentrations, higherproportion of IgG and higher avidity of Hib-ab than the control or the Hib-PS group, suggesting persisting immunological memory in aHib-c group. A mathematical model, including memory, predicted accurately the Hib-ab concentrations, which are maintained throughanamnestic responses to intervening stimuli (Hib or cross-reacting bacteria). 2003 Published by Elsevier Ltd.

Keywords: Haemophilus influenzae type b; Duration of immunity; Modelling

1. Introduction

Conjugate vaccines, in which the immunising potentialof bacterial cell surface polysaccharides (PS) has been en-hanced by covalent coupling to protein, are important newtools in prevention of infectious diseases. The first suchvaccine, the Haemophilus influenzae type b (Hib) conjugatevaccine has resulted in virtual elimination of invasive Hibdisease in the countries that have included it in their infantimmunisation programs [14]. A large amount of knowledgeof the special features associated with the novel vaccinetype, combining properties of polysaccharide and proteinantigens, has been obtained through the Hib model, i.e.clinical trials and subsequent large-scale use of the vaccine.

Recently, two further conjugate vaccines have been li-censed. Together, these vaccines against the three foremost

Corresponding author. Tel.: +358-9-47441; fax: +358-9-47448599. E-mail address: [email protected] (H. Kyhty).1 Present address: Aventis Pasteur, 2 avenue Pont Pasteur, F69367 Lyon

Cedex 07, France.

bacterial causes of acute respiratory infections and menin-gitis, Hib, Streptococcus pneumoniae and Neisseria menin-gitidis, could have a major impact on the disease burden inboth developing and industrial countries. A thorough under-standing of the rules and limitations of protective immunityelicited by the conjugate vaccines would be important inpreparation for their extensive worldwide use. In the presentarticle, we address a key question, i.e. the duration of im-munity and the consequent need of revaccination in the Hibmodel.

In the unvaccinated population serum antibodies to HibPS develop from approximately 2 years of age on in responseto subclinical infections (carriage) by Hib and other bacte-ria that have cross-reacting PS structures [58]. A plateauof approximately 12g/ml is sustained through adulthood[1], presumably in response to similar random encounterswith the Hib antigen. In this setting, the antibody concen-tration predicting protection was identified as 0.15g/ml[1,9,10] and a mathematical model constructed to predictantibody persistence after an initial response to Hib PS[11,12].

0264-410X/$ see front matter 2003 Published by Elsevier Ltd.doi:10.1016/S0264-410X(03)00524-3


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The response to the conjugate vaccine differs from theabove-described response to Hib PS in three aspects: an an-tibody response is obtained earlier, the response is higher,and the responses to later antigenic stimuli are likely to beboth higher and more rapid anamnestic responses because ofthe immunological memory elicited [1316]. To learn how

these features, and especially the immunological memoryaffect the duration of immunity we studied 910-year-oldchildren who had participated 8 years earlier in immuno-genicity studies with Hib conjugate (Hib-c) or PS vaccines.Their anti-Hib concentration was compared to the concen-tration in control children who had never received Hib vac-cine. Then the three groups of children were given a doseof Hib PS as a test vaccine for immunological memory andtheir responses assessed for antibody quantity, isotype andavidity. Furthermore, the mathematical model of antibodypersistence after Hib PS immunisation was modified to takeinto account the special features associated with conjugatevaccines.

2. Materials and methods

2.1. Subjects

All children were recruited to the study during 19931994at the Health Centre of Joensuu, Finland. The study proto-col was approved by the Ethics Committee of the NationalPublic Health Institute, Helsinki, Finland.

2.1.1. The Hib conjugate vaccine groupThirty-seven children (mean age 9.5 years, range

9.010.5) had participated in early immunogenicity studies[16,17] in the same location and received the Hib conjugatevaccine PRP-D (Connaught Laboratories Inc., Swiftwater,PA) as four injections between the ages of 3 and 18 months.

2.1.2. The control groupThirty-nine children (mean age on recruitment 9.8, range

9.010.6) had not received any Hib vaccine previously.

2.1.3. The Hib-PS vaccine groupThirteen children from a previous study [16], who had

received Hib-PS vaccine (10g of Hib PS per dose, lot7-1391-002; Lederle Laboratories, Pearl River, NY or 25gof Hib PS per dose, lot 3799, Connaught Laboratories Inc.)as four injections between the ages of 3 and 18 months wererecruited to this study at the mean age of 10.4 years (range10.310.6 years).

2.2. Samples and test vaccination with Hib PS

A venous blood sample was drawn from all children onrecruitment. After this the children were vaccinated withthe Hib PS vaccine PRP (25 g/0.5ml). The vaccine (lot B)was prepared by Wyeth Lederle Paediatrics and Vaccines,Rochester, NY, described under FDA PLA no 83-362 and

received from Dr. Pamela McInnes, Food and Drug Admin-istration, Bethesda, MD. No other vaccines were given at thesame time. A second blood sample was drawn 4 weeks later.The serum was separated and stored in aliquots at 20 Cuntil use.

2.3. Antibody assays and previous data

Total anti-Hib PS antibody concentrations were deter-mined by radioimmunoassay (RIA) [18]. The same assayand the same reference serum (lot 1983) received fromCentre for Biological Evaluation & Research (CBER),Bethesda, MD, that contained 70g/ml of anti-Hib PShas been used when the antibody response to the primaryimmunisations was determined. Furthermore, the same inhouse control sera were used throughout the present andthe previous studies. From those children that had receivedHib vaccines in early childhood, we had antibody data fromsamples taken up to 2 years of age [16,17], unpublished. In

addition, we had antibody data from our previous studiesof unimmunised children at the same ages [1619].

Immunoglobulin class specific antibody concentrationswere determined by enzyme immunoassay (EIA) as de-scribed [16] with minor modifications using Hib oligosac-charides conjugated to human serum albumin (HbOHA) [20]received from Porter Anderson (University of Rochester,Rochester, NY) as coating antigen. The two-tailed Studentst-test was used to test the significance of differences be-tween antibody concentrations in different study groups.

Avidity of anti-Hib PS antibodies was measured as de-scribed by Goldblatt et al. [21] with a modification described

by Anttila et al. [22]. In short, wells of polystyrene plates(Costar, Cambridge, MA) were coated with HbOHA [20].Low avidity antibodies were detached by 0.2M sodiumthiocyanate and the percentage of antibody left after thistreatment was calculated as relative avidity index.

2.4. Mathematical model

We formulated a model for the decline of anti-Hib PSantibody concentrations after Hib-c vaccination that tookinto account the initial response, the subsequent decay ofconcentration, and the possible restimulation due to en-counters with Hib or cross-reacting bacteria. The modelfor a polysaccharide-type response to vaccine and naturalantigenic stimuli has been described by Auranen et al. [11].The implications of this model have been further investi-gated by Leino et al. [12]. In the present study, we modifiedthe two key parameters. First, the response to vaccine (nowresponse to the fourth dose of Hib conjugate vaccine) wasestimated from observed responses to Hib conjugate vac-cine at 1.5 years of age (N= 113, estimated mean response45g/ml, 95% of responses within interval 4508g/ml,unpublished data). Second, the same log-normal distributionwas assumed to characterise the anamnestic, memory typeresponses to subsequent antigenic stimuli. These stimuli


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were taken to occur at a rate of 0.25 per year at randomencounters with Hib or cross-reacting bacteria [11].

3. Results

The life history of anti-Hib antibodies in the serum of the37 Hib conjugate vaccine recipients (Hib-c group) (Fig. 1)shows the transplacentally acquired antibodies rapidly de-clining in the first months of life, the response to the pri-mary series of three Hib-c injections given between 3 and 7months, the subsequent decline to the age of 1.5 years, fol-lowed by a strong response to the booster dose of vaccine,and again a decline in the next 6 months. All this corre-sponds to previously described responses to Hib conjugatevaccines. The new data show that in the subsequent years,up to 8 years from the last vaccine dose, there was negli-gible further decrease of the mean serum anti-Hib concen-tration, which thus remained at 3.4g/ml. The figure alsoshows the development of natural immunity to Hib in thecontrol group of children who had never received Hib vac-cine (an increase of the mean anti-Hib concentration from0.16g/ml at 2 years to 0.94g/ml at 910 years). Thus,the Hib conjugate vaccine recipients had at 910 years ofage 3.6-fold more (P < 0.001) antibodies to Hib PS than thecontrol group. The antibody was mainly IgG in both groups,and the mean avidity index 51.1 in the Hib-c group and 45.9in the control group (Table 1).

The antibody concentrations at 910 years of age in the 37Hib-c group children varied widely from 0.1 to 100 g/mlas shown by Fig. 2 in which they are plotted against the

antibody levels of the same children at 2 years of age. Of

0.01

0.1

1

10

100

0 1 2 3 4 5 6 7 8 9 10

Age, years

Anti-Hib,ug/ml

Fig. 1. The development from birth to 10 years of serum antibodies to Hib PS in children who have received four doses of the Hib conjugate vaccinePRP-D between 3 and 18 months of age (solid line) or no Hib vaccine (dotted line), based on data from the authors laboratory ( [1619], present study,unpublished).

0.01

0.1

1

10

100

0.01 0.1 1 10 100 1000

Anti-Hib at 24 or 36 mo, ug/ml

Anti-Hibat9t

o10yr,ug/ml

Fig. 2. Comparison of concentrations (g/ml) of anti-Hib PS at the age of2 years (X-axis) and at 910 years (Y-axis) in sera of children immunisedwith four doses of Hib conjugate vaccine PRP-D between the ages of 3

and 18 months. Children with >4-fold higher concentration at 910 yearsthan at 2 years are indicated with an open square.

note is that in six children the antibody concentrations at910 years were 310 times higher than at 2 years, stronglysuggesting an intervening antigenic stimulus. However, asthe stimuli themselves were not observed it was not possibleto tell from these data whether or not the response was ananamnestic response indicative of memory.

To this end, we used a different approach, i.e. giving atest dose of Hib PS to the Hib-c group of children and com-paring their responses to the responses in the control groupof children who had not received a Hib vaccine before. The

findings were consistent with immunologic memory in the


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Table 1Serum antibodies to Haemophilus influenzae type b (Hib) polysaccharide (PS) in 910 years old children before or 1 month after a test dose of Hib PSvaccine

Before test dose of Hib PS After test dose of Hib PS

No Hib Hib-c No Hib Hib-c

Anti-Hib PS (N) 39 37 39 36

IgG 0.63 (0.460.87) 5.76a (4.697.08) 28.6 (23.335.0) 107.7a (88.2131.4)IgM 0.15 (0.130.17) 0.16 (0.150.18) 1.57 (1.231.99) 0.60b (0.500.72)IgA 0.13 (0.120.15) 0.19 (0.150.23) 3.02 (2.393.81) 4.35 (3.515.40)

Relative avidity (N)d 27 34 27 34Avidity index 45.9 (37.953.9) 51.1 (44.657.5) 41.2 (34.148.3) 50.5c (45.156.0)

The children had either never received Hib vaccine (No Hib) or received (Hib-c) the Hib conjugate vaccine PRP-D as four doses between the agesof 3 and 18 months. The antibody concentrations (g/ml) were measured by enzyme immunoassay (EIA) and given as the mean (95% CI). The relativeavidity of the IgG was measured by thiocyanate EIA and is given as the avidity index.

a no Hib.b


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response-based model (panel A), and the modified, anamnes-tic response-based model (panel B). It is evident that the PSmodel gave too low predicted values in comparison to theobserved GMC (3.4g/ml; marked by arrowhead) whereasthe fit under the conjugate model in panel B was excellent.

4. Discussion

We found that the anti-Hib concentrations in 910 yearsold children 8 years after Hib conjugate vaccination were3.6-fold greater than in children who had never receivedHib vaccine. The persistence of the anti-Hib concentrationswas much longer than after vaccination with Hib PS vac-cine; the 13 children who had received Hib PS in infancythat we could trace 8 years later had antibody concentrationsnot differing from the GMC in previously unvaccinated chil-dren. This finding supports previous data from a 4.5-yearfollow-up study in Sweden [23] and 3-year follow-up stud-

ies in UK and Canada [24,25]. The antibody concentrationsreported in the Swedish study (2.1g/ml) at 6 years, andin Canada (2.2g/ml) at 45 years are rather close to the3.4g/ml found in this study at 10 years of age althoughthe conjugate vaccines used differed (PRP-T in Sweden andCanada, the generally less immunogenic PRP-D in Finland[19]).

In our study and the study from Canada with the verysatisfactory long-term results, the conjugate was given asthree doses in infancy, followed by a booster dose in thesecond year of life. By contrast, in Sweden only three dosesof PRP-T between 3 and 12 months gave a good result [23].

Less satisfactory results have been seen 23 years after onedose of PRP-D given at 19 months of age in Canada (GMC0.7g/ml [25]), after four doses of PRP-T in The Nether-lands (GMC 0.8g/ml [26]) and 3 and 4 years after thesame vaccine given in the UK program at 2, 3, and 4 monthsof age (1.06 and 0.89g/ml, respectively [24]). Whetherthe different schedules will suffice to provide sustained pro-tection from disease and carriage in the long run remains tobe seen.

We also found long-term persistence of a high, IgG dom-inated response to Hib PS vaccine in the recipients of theHib conjugate vaccine compared to children who previouslyhad received only Hib PS or no Hib vaccine. A similar testfor persistence of memory has been reported from the UK 3years after vaccination with PRP-T at 2, 3 and 4 months ofage [24]. The higher relative avidity of IgG antibody evokedin the present study by the Hib PS 8 years after vaccinationwith the conjugate vaccine further supports the concept ofan anamnestic response and long persistence of memory.

In the present study, we furthermore showed that theresponse to intervening unobserved stimuli is an essentialelement in the persistence of elevated serum antibody con-centrations after Hib conjugate vaccinessee examples inFig. 2. Mathematical modelling has provided additional ev-idence of the role of restimulation in persistence of anti-Hib

PS antibodies [11,12]. The model was initially developed forantibodies stimulated by Hib PS vaccine, but was modifiedin the present study to accommodate stronger anamnesticresponses to intervening stimuli during the 8 years followup after Hib-c vaccination. A comparison of the predictedantibody concentrations at 10 years of age with the observed

value showed that this modification was essential. An un-certainty in these calculations was the value of the force ofinfection, i.e. the rate of encounters with antigenic stimulipreviously estimated as 0.25 per year [27]. On one hand,this level might be an over-estimate for the present studyas the prevalence of Hib carriage in the study populationmay have decreased due to herd immunity generated by theintroduction of Hib-c vaccine in Finland during the 8 yearsfollow-up time of this study [2]. On the other hand, thereis no evidence suggesting that the frequency of stimuli dueto the cross-reactive bacteria would have decreased. In fact,the observed anti-Hib concentrations (0.94 g/ml) in the910-year-old children in this study who had not received

Hib vaccine was similar to adult levels before the conjugateimmunisation era, strongly arguing for continued antigenicstimuli in the population.

In conclusion, we have shown that immunity to Hib canpersist for at least 8 years after vaccination with Hib conju-gate vaccine in infancy. The persistence depends on repeatedantigenic stimuli provided by both Hib and cross-reactingbacteria, and the strong anamnestic responses to these stim-uli due to long-lasting immunologic memory. The sameelements are likely to play a role in persistence of immunityelicited by both pneumococcal and meningococcal vaccines.It would therefore be wise to collect data on their magni-

tude as a basis of a rational design of future vaccinationprograms.

Acknowledgements

We thank Teija Jaakkola and Sirkka-Liisa Wahlman fortechnical assistance in the antibody assays, Pirjo-RiittaSaranp and Minna Huittinen for recruiting, taking bloodsamples and vaccinating the children. Dr. Pamela McInnes(NIH, Bethesda, MD) provided the Hib vaccine for thestudy.

References

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